Field
This disclosure is generally with the purification of safe and efficacious active protein products in which a viral inactivation step precedes a final active protein purification step. By reversing the conventional sequence of steps of purification followed by viral deactivation, it is possible to remove inactive or denatured protein resulting from the relatively harsh conditions of the viral inactivation step. The invention is illustrated with a specific active protein known as antithrombin. As described below, however (the disclosed method is applicable to other biologically active proteins.
Antithrombin, also known as antithrombin III, AT-III or heparin cofactor, is a plasma protein with the ability to inhibit the clotting process. Antithrombin is an inhibitor of coagulation proteases whose inhibitory activity is markedly enhanced in the presence of heparin. Heparin is a sulfated glycosaminoglycan of animal origin widely used as a clinical anticoagulant.
Individuals who lack normal circulating levels of antithrombin in their blood plasma have been shown to be at increased risk of thrombosis. Deficiency states may be either hereditary or acquired. Antithrombin levels below 70% of that of pooled normal plasma are associated with thrombotic risk. Replacement therapy with purified plasma-derived antithrombin may be of considerable benefit to individuals with such deficiencies.
The enhancement of antithrombin activity by heparin is due primarily to a binding interaction between heparin and the inhibitor. The recognition of the tight and highly specific nature of this binding interaction prompted the use of immobilized heparin as an affinity support for the adsorption of antithrombin from biological fluids. See U.S. Pat. No. 3,842,061 to Anderson et al. This technique has been shown by numerous investigators to be a highly-effective step for achieving significant purification of antithrombin. Virtually all large-scale processes for the isolation of antithrombin therefore employ affinity adsorption on immobilized heparin. Other examples of antithrombin purification are well known.
However, the use of heparin affinity chromatography for direct adsorption of antithrombin from plasma at an early point in the commercial Cohn cold ethanol process is complicated for several reasons. Plasma is a highly complex mixture of proteins and other components with varying affinities for heparin. Direct contact of immobilized heparin supports with plasma results in the adsorption of many of these components. Since several different protein products are obtained from the same source plasma, serious regulatory concerns exist regarding the potential for introducing deleterious changes in these products as a result of the affinity contact step.
The adsorption of multiple plasma components on the affinity gel also requires the selective desorption of undesired contaminants before elution of the antithrombin itself. This has proved to be quite difficult to accomplish in a single affinity chromatography step and often requires the inclusion of additional purification steps to remove these contaminants. Alternatively, inclusion of extensive wash steps prior to elution of antithrombin from the heparin gel may increase purity to acceptable levels but only at a considerable cost of antithrombin yield.
Because of the large volumes usually employed during commercial plasma fractionation and the possible impact of chromatography steps on products derived from later steps of the processing, unused waste fractions of the processing have been considered as a source of antithrombin. In particular, plasma source Cohn Fraction IV-1, a normally discarded precipitate deriving from an intermediate step prior to albumin purification, has been found to be a rich source of antithrombin. See Wickerhauser et al, Vox Sang. 36, 281-293 (1979). Such a source, however, provides a non-ideal solution for chromatography (after resuspension) due to the presence of large amounts of lipoproteins and other denatured components. It is usually very difficult to obtain high levels of antithrombin purity by direct chromatography of Fraction IV-1 solutions on immobilized heparin without large sacrifices of yield.
An additional consideration for the purification of any protein from pooled blood plasma or other sources for biologically active proteins is the possibility of viral contamination. Hepatitis B virus and AIDS virus are of particular concern. With regard to Hepatitis B, it was shown that heating at 60.degree. C. for 10 hours in the presence of 0.5M sodium citrate resulted in the complete inactivation of the virus. See Tabor et al, Thrombosis Research 22, 233 -238 (1981). The majority of antithrombin activity is maintained during this heat treatment although significant and varying amounts of inactivation have been observed to occur. See Tabor et al, above, and Barrowcliffe et al, Fr. J. Haematology, 55, 37-46 (1983). Thus, the need to insure safety from virus contamination is accompanied by some risk of causing the inactivation of antithrombin itself. This latter possibility is troublesome since the injection of heat-denatured proteins may elicit a response in the patient due to the potentially neo-antigenic nature of these materials.
Until now large scale preparation methods for clinical antithrombin concentrates have incorporated a pasteurization step subsequent to the affinity adsorption step on immobilized heparin. Thus, these products potentially contain large amounts of denatured antithrombin as shown by Barrowcliffe et al, above. We are unaware of methods of preparing biologically active protein products in which denatured or inactive proteins and other impurities are removed subsequent to a viral inactivation step.